Caveolae (CAV) are novel sub-cellular transport vesicles postulated to be involved in multiple cellular processes with demonstrated pathophysiological and clinical relevance. Although an important role in signal transduction was initially ascribed to CAV, its relevance in this context has been recently underlined by the proposal of a new term to describe this organelle (i.e. signalosomes or transducisomes). Furthermore, several key components of the signal transduction pathways, postulated to be involved in many cells in the expression of a "normal" or abnormal differentiated phenotype, have been linked to or proposed to be regulated by CAV and its primary component caveolins. Recently, we have established the expression of all caveolin isoforms in C6 glial cell line and suggested a possible role for CAV in differentiation of this model system and PC12 cells. Subsequently, at this time we are proposing to test our new working hypothesis that transient up and down regulations of both caveolin-1 and -2 isoforms are required for nerve growth factor (NGF) induced PC12 cells differentiation. Testing of this novel hypothesis will require a more detailed characterization of the changes in caveolin/caveolae dynamics and function associated with the differentiation process, followed by a precise and controlled manipulation of the expression of these two isoforms by employing molecular biology tools. In this context the following specific aims are proposed: Specific aim 1: To continue with the systematic determination of changes in the expression of caveolin-1 and -2 isoforms, and caveolae biogenesis and dynamics, associated with PC12 cells differentiation. Specific Aim 2: To up-regulate the expression of caveolin-1 and -2 isoforms at different times during the differentiation process by using stable transfectants that can expression caveolin-GFP chimeras in a tetracycline regulated system. Specific aim 3: To down-regulate the expression of caveolin-1 and -2 isoforms at different times during the differentiation process by using stable transfectants that can co-express GFP and caveolin-1 or -2 anti-sense in a tetracycline regulated system. This aim will be achieved by a combination of complementary methods including Western Blot analysis of several cell fractions and immunofluorescent studies for 3-D image reconstruction analysis of caveolin isoforms localization and co-localization with signal transduction elements. This analysis will be made in the context of the transition to the differentiated phenotype, as determined by morphological changes and the expression of specific differentiation markers.